1. Field of the Invention
The present invention relates to [21-leucine] human urogastrone (often referred to as "[21-L] hUG" hereinafter) and a process for the production of [21-L] hUG by genetic engineering. More specifically, the present invention further includes a chemically synthesized structural gene for [21-L] hUG, a corresponding recombinant plasmid and a transformed cell.
2. Discussion of the Backgroud
Human urogastrone (hUG) is a peptide comprising 53 amino acid residues and having an action of inhibiting gastric acid secretion, which was discovered in human urine by Gregory et al in 1975 [Nature, 257, 325 (1975)].
hUG is a polypeptide having the following structure and contains three disulfide bonds in the molecule: EQU H-Asn-Ser-Asp-Ser-Glu-Cys-Pro-Leu-Ser-His-Asp-Gly-Tyr-Cys-Leu-His-Asp-Gly-V al-Cys-Met-Tyr-Ile-Glu-Ala-Leu-Asp-Lys-Tyr-Ala-Cys-Asn-Cys-Val-Val-Gly-Tyr- Ile-Gly-Glu-Arg-Cys-Gln-Tyr-Arg-Asp-Leu-Lys-Trp-Trp-Glu-Leu-Arg-OH
In the same year, Cohen et al discovered a human epidermal growth factor (hEGF), which is a growth factor promoting proliferation and keratinization of the epithelial tissue, in human urine and isolated and purified this factor [Proc. Natl. Acad. Sci. USA, 72, 1317 (1975)]. The entire amino acid structure of hEGF has not been elucidated, but according to recent researches on the basis of gel electrophoretic analysis and receptor competition with mouse epidermal growth factor (mEGF), it is now considered that hEGF and hUG are identical.
It is now confirmed that hUG and hEGF have common physiological actions [Ann. Rev. Biochem., 48, 193 (1979)]. Furthermore, by comparison in view of the physiological actions of hUG with mEGF having the same 37 amino acid residues as those of hUG among the 53 amino acid residues and three S-S bonds at the same positions as in hUG, the activity of hUG/hEGF or mEGF has been found beyond species, and it is therefore considered that these physiological actions are due to the specific stable three-dimensional structure involved.
However, this hUG (hEGF) is isolated only in a minute amount from human urine, and mass production has been desired. Recent developments in genetic engineering have produced excellent results in the production of heterologous proteins by E.coli or the like. Accordingly, application of this technique to the mass production of hUG is naturally considered and has already been proposed (Japanese Patent Application Laid-Open Specification No. 122096/82).
As one most effective method for synthesizing peptides according to the genetic engineering process, mention can be made of a process in which: a gene coding for an intended peptide is linked to a structural gene for another protein derived from a host (such as .beta.-galactosidase or TrpE of E. coli through AUG (a codon for methionine) and is cloned in the host; the biosynthesized, so-called fused protein is treated with cyanogen bromide; and the intended peptide is recovered (Japanese Patent Application Laid-Open Specifications No.84603/81 and No.145221/81). However, synthesis of a peptide containing Met cannot be performed according to this process. Since hUG contains Met as its 21st amino acid residue from the N-terminus, the above process cannot be applied directly to synthesis of hUG. For example, the above mentioned laid-open specification (Japanese Patent Application Laid-Open Specification No.122096/82) discloses a process in which by utilizing a trypsin-insensitive property of hUG, the objective peptide is produced in the form of a fused protein having Lys-Lys attached upstream of the N-terminus and the intended peptide is recovered by trypsin treatment. However, according to another report [Journal of Japanese Medical Association, 85, 833 (1981)], the activity of hUG is lost if five amino acids of hUG are removed from the C-terminus. Accordingly, it is doubtful whether or not hUG is cleaved at the desired point. Therefore, it is not deemed that this process will be advantageous.
Based on the supposition that the 21st amino acid residue, namely, Met, of hUG does not participate in the physiological activity of hUG, we conducted research and confirmed that when Met is substituted by Leu (leucine) having a similar structure so that a conventional cyanogen bromide treatment of a fused protein is applicable, the recovered [21-L] hUG has an activity equivalent to the activity of hUG (hEGF). The present invention is based on this and other findings.
Although it has been clarified that the activity of hUG (hEGF) depends on the three-dimensional structure having three disulfide bonds, its specific active site and mechanism of its action are not clear at present. Accordingly, the above technical concept is not believed obvious to those skilled in the art.